The attorney general’s office said Antonio Ledezma, 59, would remain in a military prison pending his trial.
President Nicolas Maduro earlier accused the opposition mayor of being involved in a US-backed coup.
This comes on the anniversary of the start of protests against Nicolas Maduro’s rule that left dozens of people dead.
On February 20, opponents of President Nicolas Maduro again protested in Caracas against what they described as a crackdown on his political opponents.
The US has also denounced the “systemic intimidation” by the Venezuelan authorities of the country’s opposition.
State department spokeswoman Jen Psaki said: “Venezuela’s problems cannot be solved by criminalizing legitimate, democratic dissent.”
The attorney general’s office said on February 20 that Antonio Ledezma was charged with conspiracy to plot violence against the government.
This came a day after camouflaged police smashed into the mayor’s office in the banking district and carried him away.
President Nicolas Maduro has said the opposition leader must answer “for all the crimes committed against the country’s peace and security”.
Venezuela’s opposition is now demanding the authorities produce any evidence of the alleged conspiracy.
Opposition leader Henrique Capriles asked: “Does Maduro think that putting everyone in prison is going to get him 50 popularity points or that he’s going to win elections?”
Antonio Ledezma was on a list of people and foreign powers named by Nicolas Maduro last week as attempting to bring down his administration.
Antonio Ledezma, Caracas mayor since 2008, replied that it was government corruption that was bringing down Venezuela.
Last year, weeks of anti-government protests in Venezuela led to more than 40 deaths.
Venezuela’s economy has been heavily affected by the drop in oil prices and in late January, thousands of people joined an opposition march in Caracas.
Venezuelans voiced dissatisfaction with high inflation, crime and the shortage of many staple goods in the shops.
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